PROJECT SUMMARY Bacteria exist as single cells in a planktonic state, but they also associate into multicellular communities known as biofilms. A rise in the intracellular levels of the second messenger cyclic diguanylate (c-di-GMP) promotes biofilm formation in a wide range of bacterial species. Thus, understanding the control of c-di-GMP levels is essential to comprehend the transition between planktonic to biofilm states. Bacteria harbor diguanylate cyclases and phosphodiesterases, which are enzymes that synthesize and degrade c-di-GMP, respectively. A given bacterial species often harbors a large repertoire of these enzymes, each regulating a distinct cellular process. We reported that a Salmonella enterica serovar Typhimurium strain lacking the mgtC virulence gene harbors increased levels of c-di-GMP and of cellulose, a major component of biofilms that inhibits Salmonella replication inside macrophages. We have now identified the specific diguanylate cyclases responsible for the c-di-GMP- dependent cellulose synthesis in the mgtC mutant and established that they undergo post-translational activation. We have also utilized a novel c-di-GMP fluorescence reporter to select mutations (currently being mapped by high-throughput sequencing) that affect the activities of these diguanylate cyclases. This proposal seeks to define how the identified diguanylate cyclases are activated, and to determine the role of MgtC in controlling crucial physiological functions in Salmonella during infection. Specifically, the identification of genes controlling these diguanylate cyclases will enable us to explore how these enzymes are controlled during Salmonella replication inside macrophages and, by proxy, make testable inferences about metabolic changes promoted by MgtC. These investigations will reveal the cues promoting c-di-GMP synthesis and biofilm formation, and how bacterial pathogens must control their metabolism to replicate in host phagocytic cells.